1. Field of the Invention
The present invention relates to a method of inhibiting glycolysis of glucose in blood samples.
In the determination of glucose, lactic acid or pyruvic acid in blood samples, if collected blood samples are allowed to stand at room temperature, glycolysis reactions with glycolytic enzymes in blood will proceed with a result that glucose is decreased and lactic acid and pyruvic acid formed by the glycolysis are increased. These reactions are known as so-called Embden-Meyerhof pathway. It has also been experimentally indicated by us that glucose in blood stored at 37.degree. C. is decreased from an initial value of 110 mg/ml to ca. 83 mg/ml after 3 hours. Usually, as time needed between the blood collection and the analytical operation is at least 2 to 3 hours (in case of analysis at hospitals) or occasionally dozens of hours (in case of analysis at other laboratories), it is required to inhibit the glycolysis reactions as described above. The method according to the invention provides means effectively used for the analysis of such blood components.
2. Description of Prior Arts
Heretofore, inhibition of glycolysis reactions in blood samples is known to be effected by (1) a method by which a glycolysis inhibitor principally consisting of a fluoride compound is added, (2) a method by which plasma and enzyme-containing blood cells are separated immediately after blood collection, (3) a method by which deproteination is carried out rapidly after blood collection or (4) a method by which blood samples after collection are stored at a low temperature. All of the methods (2), (3) and (4), however, are troublesome in handling of samples immediately after blood collection or transport and storage of blood samples. The method (1) is widely employed, because the fluoride compound used therein such as, for example, sodium fluoride (NaF) or potassium fluoride (KF) is believed to be a specific inhibitor of enolase (an enzyme) which acts in the course of Embden-Meyerhof glycolysis pathway and is used in the form of a very small amount of powders placed in advance in a sample-collecting vessel for easy mixing with blood sample. A blood-collecting tube widely employed in practice for this purpose is VENOJECT (FH) (tradename of a product manufactured by Terumo K.K.) in which powders principally consisting of NaF are placed. In this method, as F ions from NaF at a higher concentration induce elution of hemoglobins from blood cells, that is, hemolysis, NaF is added in an amount to produce a concentration as low as 1.25 mg/ ml or below.
Though many studies have been made on the optimal conditions for the use of fluorine compounds as a glycolysis inhibitor, there remains questions as to whether fluoride compounds are satisfactory with respect to time required for onset of the glycolysis-inhibiting action as well as to duration of the glycolysis-inhibiting action. As shown by line (2) in FIG. 1 of the attached drawing, it has experimentally been demonstrated by us that the glucose level in blood to which 1.25 mg/ml (blood) of NaF (corresponding to the amount incorporated in VENOJECT (FH) manufactured by Terumo K.K. cited above) has been added is reduced by ca. 6% in 1 hour and by ca. 8% in 3 hours. The reduction to such degrees is pointed out also by Tatsuo Nagashima and others in an article entitled "Glycolysis inhibition in blood glucose determination " appearing in Japanese Journal of Medical Technology (special volume for the 33rd Congress of Japanese Association of Medical Technologist, Vol. 33, No. 3, published March 25, 1984) p. 463. As described above, glycolysis inhibition by means of the prior-art glycolysis inhibitors is questionable regarding rapid onset and duration of the action. It has been confirmed by ion chromatographic analysis that F and I ions such as those from the fluoride compounds widely used as the glycolysis inhibitor and monoiodoacetic acid are easily and rapidly passed through the cell membrane of red blood cell to be introduced in the cell in which glycolytic enzymes are present. Therefore, longer time required for onset of the action of the prior-art glycolysis inhibition is believed not due to their cell-membrane permeability but to the fact that the enzyme (enolase) on which these inhibitors act is not a rate-determining enzyme for the glycolysis system.